Advantage of urine based molecular diagnosis of Zika virus

International Urology and Nephrology, Dec 2016

Zika virus (ZIKV) infection is an emerging global health concern, and diagnostic recommendations are currently developing based on new information. Several case or small sample size studies using both urine and blood samples suggest that ZIKV RNA can be detected at higher levels and for a longer time after onset of infection in urine compared to blood. We recommend that urine and serum collection for molecular testing be a standard part of evaluating patients for ZIKV infection, and that urine is a good alternative testing sample when blood collection is problematic.

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Advantage of urine based molecular diagnosis of Zika virus

Int Urol Nephrol Advantage of urine based molecular diagnosis of Zika virus Laura E. Lamb 0 1 2 3 4 5 Sarah N. Bartolone 0 1 2 3 4 5 Sebla B. Kutluay 0 1 2 3 4 5 Daniela Robledo 0 1 2 3 4 5 Alexandra Porras 0 1 2 3 4 5 Mauricio Plata 0 1 2 3 4 5 Michael B. Chancellor 0 1 2 3 4 5 0 Department of Molecular Microbiology, Washington University in St. Louis , St. Louis, MO , USA 1 Department of Urology, Oakland University William Beaumont School of Medicine , Royal Oak, MI , USA 2 Department of Urology, William Beaumont Hospital , 3811 West 13 Mile Road, Suite 187, Royal Oak, MI 48073 , USA 3 Subdireccion de Estudios Clínicos, Fundación Santa Fe de Bogotá University Hospital , Bogotá D.C. , Colombia 4 School of Medicine, Universidad de Los Andes , Bogotá D.C. , Colombia 5 Department of Urology, Fundación Santa Fe de Bogotá University Hospital , Bogotá D.C. , Colombia Zika virus (ZIKV) infection is an emerging global health concern, and diagnostic recommendations are currently developing based on new information. Several case or small sample size studies using both urine and blood samples suggest that ZIKV RNA can be detected at higher levels and for a longer time after onset of infection in urine compared to blood. We recommend that urine and serum collection for molecular testing be a standard part of evaluating patients for ZIKV infection, and that urine is a good alternative testing sample when blood collection is problematic. Zika virus; Urine biomarker; Molecular diagnostic; Zika infection - Introduction Infection with Zika virus (ZIKV) is of growing concern since it is suspected with causing brain defects in newborns Methods * Laura E. Lamb Table 1 Detection of ZIKV RNA in various sample types (adopted from [42]) Detection (days) after symptom onset Viral RNA load (copies/mL) Cerebrospinal fluid Not applicable RealStar® Zika virus RT-PCR Kit (available from Altona Diagnostics) and the Zika virus RNA Qualitative RealTime RT-PCR (available from Quest Diagnostic Tests). There are currently several diagnostic tests available for ZIKV. Quantitative reverse transcription PCR (RT-PCR) of ZIKV RNA in serum or plasma samples is the primary approach. However, this method can give a negative result as soon as 3–5 days after symptom onset [8, 9], which does not exclude ZIKV infection. IgM antibody detection by ELISA can be done 4 days after onset but cannot distinguish between ZIKV from Dengue Fever virus, Yellow Fever virus, and possibly to vaccines against flaviviruses [8, 10–12]. Plaque-reduction neutralization test (PRNT) can measure virus-specific antibodies, but cross-reactivity also exists. Advantages of non‑invasive sample collections Urine, saliva, nasopharygeal, and/or buccal (cheek swab) testing are non-invasive and are an attractive sample for diagnostic testing in which blood collection can be difficult. This may include young children, neonates, elderly, patients with hemorrhagic syndromes, patient refusal, or patients who present with small, dehydrated, or elusive veins. Sample collection can also be done at field locations where trained medical personal or facilities may be lacking, and allow for self-collection within communities for surveillance or epidemiology studies during an outbreak. Lastly, ZIKV has been reported to be transmitted through blood donation [13]. Diagnosis of other viral infections in urine and saliva samples has been reported for human immunodeficiency virus (HIV), Hepatitis A, B, and C, and rubella [14–20], and more recently in other flaviviruses, West-Nile virus (WNV) [21–23] and Dengue (DENV) serotypes -1, -2, -3, and -4 [24–35]. WNV can be detected longer in urine than serum [21] and can be isolated from urine [22, 23]. Some 1 3 studies also suggest that like WNV, DENV can be detected for a longer time in urine than saliva or serum [25, 29, 31, 32]. Urine may have a longer detection window for molecular diagnosis than serum or plasma samples Kutsuna and colleagues first reported that ZIKV RNA could be detected by RT-PCR in the urine but not the serum of an infected patient [36]. ZIKV has also been reported to be detected in urine of adults [37–39] and at least one neonate [37]. It is also detectable in the amniotic fluid (comprising mostly of fetal urine) of fetuses with microcephaly or fetal brain abnormalities [40, 41]. The diagnostic utility of urine was best characterized in a more extensive study of six infected patients from a 2014 ZIKV outbreak in New Caledonia, from which urine and serum from the same patients at multiple comparable time points were investigated [38]. Importantly, ZIKV is detectable for a longer time frame post-infection in urine than serum (7 or more days after becoming undetectable in serum) [38] and at a higher viral load in urine (up to 220 × 106 copies/mL) than in corresponding serum samples (up to 8.1 × 106 copies/ mL) [42]. Other groups have also reported either a longer detection window or an increased ZIKV detection in urine over other bodily fluids on a case study basis (Tables 1 and 2) [30, 37, 43–46], although there have been cases where ZIKV was detected in the serum but not urine [47]. A larger cohort of paired serum and urine samples would be beneficial to support these findings. ZIKV was even detected in urine up to 15–21 days after onset of Guillian–Barré syndrome in two patients; ZIKV was not detectable in plasma or CSF for either patient and detection may not be related to neurological symptoms [45]. Isolation and sequencing of ZIKV from urine samples has also been documented [39, 44, 48]. The Centers for Disease Control and Prevention (CDC) now recommends that urine samples be collected am m in e te V eo nd s su V tiiv ed IK sac lia a am 2 en 24 la 75 ra 12 e 20 t C ID n 4 t 7 a 2 y 9 e 58 . o 9 e 6 h 8 u 4 o 2 M PM rh 76 zé 69 ino 67 san 269 ssu 562 e ( o 2 o 2 h 2 d K R S M M D ID 1 3 <14 says after onset of symptoms in patients with suspected ZIKV infection and that molecular testing of urine be performed in conjunction with serum testing [49]. The CDC Trioplex rRT-PCR assay is the only ZIKV diagnostic test authorized by the FDA for urine. It is important to note that urine may not work as well as blood samples for assays other than RT-PCR, including IgM assays. Lastly, the majority of studies used only the primers described in Lanciotti et al. [12]. Other non‑invasive samples ZIKV has also been reported to be detected in saliva of both neonates and adults [37, 39, 50] and ZIKV can be isolated from saliva [39]. In a study comparing paired saliva and serum samples (n = 182) from the 2013–2014 French Polynesia outbreak, saliva had increased rate of molecular detection but not increased window of detection [50]. The authors noted that ZIKV RNA could be negative in some saliva samples while still positive in blood samples, and since blood samples are required for other laboratory tests that saliva could not act as a replacement. ZIKV has also been detected in semen of a few men, and supports the idea that ZIKV can be spread by sexual transmission [5, 51–53]. In the few cases ZIKV has been evaluated in semen, the infectious load was considerably high and persisted over 8–10 weeks past symptom onset [5, 51–53]. Viral load was several logs of magnitude higher than corresponding urine samples, and not detectable at all in serum. This area merits more investigation, especially given the potential for ZIKV sexual transmission and poor pregnancy outcomes, but may be a good option for late diagnosis in men. Implications for policy and practice Taken together these studies suggests that urine samples should be collected in addition to blood for molecular testing of ZIKV especially if samples are not collected within first few days of symptom onset. This may result in an increased number of laboratory confirmed cases. Urine samples may also allow for easier monitoring of potentially exposed individuals who are at high risk for ZIKV infection complications, such as pregnant women, or individuals were collection of blood is problematic. Lastly, molecular testing of urine samples may aid in large epidemiological or surveillance studies. 1 3 Several studies have now been published using paired urine and serum or plasma samples, suggesting that overall, ZIKV RNA can be detected at higher levels and for a longer time after onset of infection in urine compared to serum. Semen may also allow late detection in men. Together, this suggests that ZIKV may be shed through the urine and may have a reservoir or even be actively replicating in the genito-urinary tract. In summary, the results of these studies suggests that urine samples should be considered for collection in addition to blood for molecular testing of ZIKV, and may result in an increased number of laboratory confirmed cases. Urine samples may also allow for easier monitoring of potentially exposed individuals, especially pregnant women, couples wanting to conceive, or individuals with suspicious symptoms. Funding This study was funded in part by the Maureen and Ronald Hirsch family philanthropic contribution. Compliance with ethical standards Conflict of interest Drs. Lamb and Chancellor have a pending provision patent on urine biomarkers using proteins and RNA in urine for infectious and other urologic diseases. For the remaining authors none were declared. No funding was received for this work. Ethical approval This article does not contain any studies with human participants and animals performed by any of the authors. Informed consent None. 1. Schnirring L ( 2015 ) Zika virus spreads to more countries . In: Center for Infectious Disease Research and Policy (ed) U. of Minnesota Academic Health Center 2. Oehler E , Watrin L , Larre P et al ( 2014 ) Zika virus infection complicated by Guillain-Barre syndrome-case report , French Polynesia, December 2013 . Euro Surveill 19 ( 9 ): 20720 3. 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Advantage of urine based molecular diagnosis of Zika virus, International Urology and Nephrology, 2016, 1961-1966, DOI: 10.1007/s11255-016-1406-9